COMPARISON BETWEEN INTRAUTERINE INSEMINATION WITH OVULATION INDUCTION VERSUS NATURAL OVULATORY CYCLE IN MALE FACTOR OF INFERTILITY

Thesis Submitted to the Faculty of Medicine Alexandria University In partial fulfillment of the requirements of the degree of

Master of Obstetrics and Gynecology

By

Mohammed Ahmed Abd El Aty Abou El Maaty Azab

MBBCh, Alex. Resident El-Shatby Maternity University Hospital Department of Obstetrics and Gynecology

Faculty of Medicine Alexandria University 2013

COMPARISON BETWEEN INTRAUTERINE INSEMINATION WITH OVULATION INDUCTION VERSUS NATURAL OVULATORY CYCLE IN MALE FACTOR OF INFERTILITY

Presented by

Mohammed Ahmed Abd El Aty Abou El Maaty Azab MBBCH. Alex for the Degree of

Master
in Obstetrics and Gynecology Examiners committee Prof. Dr. . Emad Abd El Meniem Darwish Professor of Obstetrics and Gynecology, Faculty of Medicine University of Alexandria Prof. Dr. Mohammed Salah El Din Abd Rabbo Professor of Obstetrics and Gynecology, Faculty of Medicine University of Alexandria Prof. Dr. Mostafa Abd El Khalik Abd Allah Atya Professor of Obstetrics and Gynecology, Faculty of Medicine University of Sohag.
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Approved
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SUPERVISORS Prof. Dr. . Emad Abd El Meniem Darwish Professor of Obstetrics and Gynecology, Faculty of Medicine, University of Alexandria. Dr. Yasser Saad El-Kassar Lecturer of Obstetrics and Gynecology, Faculty of Medicine, University of Alexandria.
.. ..

ACKNOWLEDGEMENTS
I wish to express my sincere gratitude and gratefulness to Prof. Dr. Emad Abd El Meniem Darwish, Professor of Obstetrics and Gynecology, Faculty of Medicine, University of Alexandria, for his kind supervision and constant encouragement . In fact, it has been a great honor to work under his supervision. I am greatly indebted and appreciating to Dr. Yasser Saad El-Kassar, lecturer of Obstetrics and Gynecology, Faculty of Medicine, University of Alexandria. His useful suggestions, generous help and hard work have made it possible to complete this work. I find no wards of appreciation for his careful hard work and help.

LIST OF CONTENTS
Chapter Page ACKNOWLEDGMENT...........................................................................i LIST OF CONTENT ............................................................................... ii LIST OF TABLES .................................................................................. iii LIST OF FIGURES ................................................................................. ii I. II. III. IV. V. VI. VII. INTRODUCTION........................................................................ 1 AIM OF THE WORK ................................................................ 11 PATIENTS ................................................................................... 12 METHODS .................................................................................. 13 RESULTS ................................................................................... 14 DISCUSSION : ............................................................................. SUMMARY ................................................................................. 42

VIII. CONCLUSIONS ......................................................................... 44 IX. X. RECOMMENDATIONS ............................................................ 45 REFERENCES ............................................................................ 46 PROTOCOL ARABIC SUMMARY

LIST OF TABLES
Table (1) (2) (3) (4) (5) Comparison between the two studied groups according to demographic data. Comparison between the two studied groups according to day 3 FSH. Comparison between the two studied groups according to male age and smoking habit. Comparison between the two studied groups according sperm parameters regarding sperm count. Comparison between the two studied groups according to sperm sperm parameters regarding sperm motility by percentage (%). Comparison between the two studied groups according to sperm morphology. Distribution of the studied cases of group II according to number of follicles at day of hCG. Distribution of the studied cases of group II according to number of ampoules of HMG used for induction of ovulation. Comparison between the two studied groups according to clinical pregnancy rate. Page 14 15 16 19 21

(6) (7) (8) (9)

22 23 25 27

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LIST OF FIGURES
Figure (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) Comparison between the two studied groups according to age. Comparison between the two studied groups according to Duration of infertility Comparison between the two studied groups according to BMI. Comparison between the two studied groups according to day 3 FSH. Comparison between the two studied groups according to male age. Comparison between the two studied groups according to smoking habit.. Comparison between the two studied groups according sperm count. Comparison between the two studied groups according to sperm motility by percentage (%). Comparison between the two studied groups according to sperm abnormal forms. Distribution of the studied cases of group II according to number of follicles at day of hCG. Distribution of the studied cases of group II according to number of ampoules of HMG used for induction of ovulation. Comparison between the two studied groups according to clinical pregnancy rate. Page 17 17 18 20 21 22 24 26 27 28 30 32

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LIST OF ABBREVIATION
BMI HSG TSH FSH PCOS LH CCCT ml mIU pg IVF mg AFC AMH TGF-beta ng HyCoSy WHO ICSI CC hCG hMG rFSH GnRH HAS HTF BWW : : : : : : : : : : : : : : : : : : : : : : : : : : : Body mass index Hystero salpingogram Thyrotropin Follicle stimulating hormone Polycystic ovary syndrome leuteinizing hormone clomiphene citrate challenge test Milli litre Milli international unit Pictogram In-vitro Fertilization Milligram Antral follicle count Anti-mullerian hormone Tissue growth factor beta Nanogram hysterosalpingo-contrast sonography world health organization intracytoplasmic sperm injection Clomiphene citrate Human chorionic gonadotropin Human menopausal gonadotropin Recombinant follicle stimulating hormone Gonadotropin releasing hormone Human serum albumin Human tubal fluid Biggers, Whitten and Whittingham

INTRODUCTION
INFERTILITY
Definition: Infertility is a unique medical condition because it involves a couple, rather than a single individual. It is defined as failure of a couple to conceive after 12 months of regular intercourse without use of contraception in women less than 35 years of age; and after six months of regular intercourse without use of contraception in women 35 years and older.(1) Fecundability, the probability of achieving a pregnancy in one menstrual cycle, is a more accurate descriptor because it recognizes varying degrees of infertility. Causes of infertility: One population-based study reported that 26 percent of cases of infertility are due to male factor (hypogonadism, post-testicular defects, seminiferous tubule dysfunction) ,21 percent due to ovulatory dysfunction,14 percent due to tubal damage ,6 percent are due to endometriosis ,6 percent are due to coital problems,3 percent are due to cervical factor and 28 percent are unexplained cause of infertility.(2) Timing of infertility evaluation: The general consensus among infertility experts is that infertility evaluation should be undertaken for couples who have not been able to conceive after 12 months of unprotected and frequent intercourse, but earlier evaluation should be undertaken based on medical history and physical findings, and in women over 35 years of age.(3,4) Some authorities have proposed initiating an infertility work-up after six months of fertility-oriented intercourse without conception since prospective cohort studies have shown that a significant decline in fecundity occurs by this time .(5,6,7) The timing of initial evaluation of infertility depends upon the age of the female partner, as well as the couple's historical risk factors . Women experience a decline in fecundity as the ovary ages, especially after age 30. (8) Significantly delaying the evaluation and treatment of an infertile woman in her mid-thirties may
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diminish the success rate once therapy is initiated. For these reasons, in women between 35 and 40 years of age, we initiate the infertility evaluation after six months of frequent unprotected intercourse without conceptionand we initiate the evaluation after less than six months in women over 40 years of age.(4) Evaluation is also initiated promptly if the female partner has a history of risk factors for premature ovarian failure (previous extensive ovarian surgery, exposure to cytotoxic drugs or pelvic radiation therapy, autoimmune disease, smoking, strong family history of early menopause/premature ovarian failure, advanced stage endometriosis, or known or suspected uterine/tubal disease .(9) Male factors can also be indications for initiating early evaluation of the male partner. These factors include a history of testicular trauma requiring treatment, adult mumps, impotence or other sexual dysfunction, chemotherapy and/or radiation, or a history of subfertility with another partner.(9)

Evaluation of female infertility

History and physical examination. History : The most important points in the history are: Duration of infertility and results of previous evaluation and therapy. Menstrual history (cycle length and characteristics), which helps in determining ovulatory status. For example, regular monthly cycles with molimina (breast tenderness, ovulatory pain, bloating) suggest that the patient is ovulatory and characteristics such as severe dysmenorrhea suggest endometriosis. Medical, surgical, and gynecological history (including sexually transmitted infections, pelvic inflammatory disease, and treatment of abnormal Pap smears) to look for conditions, procedures, or medications potentially associated with infertility. At a minimum, the review of systems should determine whether the patient has symptoms of thyroid disease, galactorrhea, hirsutism, pelvic or abdominal pain, dysmenorrhea, or dyspareunia. Young women who have undergone unilateral oophorectomy generally do not have reduced fertility since young women have many primordial follicles
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per ovary; however, prior unilateral oophorectomy may impact fertility in older women as they may develop diminished ovarian reserve sooner than women with two ovaries. (10) Obstetrical history to assess for events potentially associated with subsequent infertility or adverse outcome in a future pregnancy. Sexual history, including sexual dysfunction and frequency of coitus. Infrequent or ineffective coitus can be an explanation for infertility. Family history, including family members with infertility, birth defects, genetic mutations, or mental retardation. Women with fragile X mutation may develop premature ovarian failure, while males may have learning problems, developmental delay, or autistic features. Personal and lifestyle history including age, occupation, exercise, stress, dieting/changes in weight, smoking, and alcohol use, all of which can affect fertility. Physical examination: The physical examination should assess for signs of potential causes of infertility. The patient's body mass index (BMI) should be calculated and fat distribution noted, as extremes of BMI are associated with reduced fertility and abdominal obesity is associated with insulin resistance. Incomplete development of secondary sexual characteristics is a sign of hypogonadotropic hypogonadism. A body build that is short and stocky, with a squarely shaped chest, suggests Turner syndrome. Abnormalities of the thyroid gland, galactorrhea, or signs of androgen excess (hirsutism, acne, male pattern baldness, virilization) suggest the presence of an endocrinopathy (eg, hyper - or hypothyroidism, hyperprolactinemia, polycystic ovary syndrome, adrenal disorder). Tenderness or masses in the adnexae or posterior cul-de-sac (pouch of Douglas) are consistent with chronic pelvic inflammatory disease or endometriosis. Palpable tender nodules in the posterior cul-de-sac, uterosacral ligaments, or rectovaginal septum are additional signs of endometriosis. Vaginal and cervical structural abnormalities or discharge suggest the presence of a mllerian anomaly, infection or cervical factor. Uterine enlargement, irregularity, or lack of mobility are signs of a uterine anomaly, leiomyoma, endometriosis, or pelvic adhesive disease. Diagnostic tests:

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In addition to the history and physical examination, the initial diagnostic evaluation consists of: Documentation of normal ovulatory function. Women with regular menses approximately every four weeks with moliminal symptoms are almost always ovulatory. A test to rule out tubal occlusion either hystero salpingogram (HSG), but laparoscopy with chromotubation may be more appropriate in women suspected of having endometriosis.

Assessment of ovulatory function:

In women who do not have grossly abnormal menstrual cycles indicative of ovulatory dysfunction, laboratory assessment of ovulation should be performed. Ovulation is most easily documented by a mid-luteal phase serum progesterone level, which should be obtained approximately one week before the expected menses. For a typical 28-day cycle, the test would be obtained on day 21. A progesterone level >3 ng/mL is evidence of recent ovulation .(11) If the progesterone concentration is <3 ng/mL, the patient is evaluated for causes of anovulation. The minimal work-up includes serum prolactin, thyrotropin (TSH), follicle-stimulating hormone (FSH), and assessment for polycystic ovary syndrome (PCOS). An alternative is to have the patient use an over-the-counter urinary ovulation prediction kit. These kits detect leuteinizing hormone (LH) and are highly effective for predicting the timing of the LH surge that reliably indicates ovulation. Home kits have a 5 to 10 percent false positive and false negative rate. Other methods of determining ovulation, such as serial ultrasounds to follow the development and ultimately the disappearance of a follicle and endometrial biopsy to document secretory changes in the endometrium are too expensive or invasive for routine diagnostic assessment of ovulation.

Assessment of ovarian reserve:

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The identification of diminished ovarian reserve is an increasingly important component of the initial infertility evaluation as patients are presenting for diagnostic evaluation later in their reproductive life span. 1. Day 3 FSH and CCCT (clomiphene citrate challenge test):

we obtain a day 3 FSH concentration and consider that: A value less than 10 mIU/mL is suggestive of adequate ovarian reserve. levels of 10to 15 mIU/ml are borderline. A level above 15 mIU/ml indicates poor reserve

Cycle day 3 estradiol level also is checked, although there are conflicting data as to whether it is predictive of ovarian reserve and the response to ovarian stimulation. (12, 13) We consider a value <80 pg/mL suggestive of adequate ovarian reserve, but other cut-offs are also utilized. In one prospective study of women undergoing IVF, day 3 estradiol levels >80 pg/mL resulted in higher cycle cancellation rates andlower pregnancy rates, and estradiol levels >100 pg/mL were associated with a 0 percent pregnancy rate . (14) If CCCT is performed, we consider FSH less than 15 mIU/mL on both day 3 and day 10 after five days of clomiphene intake suggestive of adequate ovarian reserve; an elevated FSH level on either day 3 or day 10 suggests decreased ovarian reserve. Estradiol can be measured on day 3, but a cycle day 10 estradiol is not part of the standard CCCT as it reflects the magnitude of the ovarian follicular response to clomiphene 100 mg daily for five days, not ovarian reserve. 2. Antral follicle count (AFC): Ultrasound examination can be used to determine the number of antral follicles (defined as follicles measuring 2 to 10 mm in diameter). On transvaginal ultrasound, the presence of four to 10 antral follicles between days two and four of a regular menstrual cycle suggests good ovarian reserve, whereas a low AFC suggests poor reserve .( 15, 16,17) 3. Anti-mullerian hormone (AMH):
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Anti-mllerian hormone (AMH) is a member of the TGF-beta family and is expressed by the small (<8 mm) preantral and early antral follicles. (18)The AMH level reflects the size of the primordial follicle pool(19); AMH is undetectable at menopause . (20) The AMH level appears to be an early, reliable, direct indicator of declining ovarian function, however, there is no consensus on the appropriate threshold value.(20-22) A serum AMH level above 0.5 ng/mL is consistent with good ovarian reserve, while lower levels suggest the presence of a depleted ovarian follicle pool. Levels less than 0.15 ng/mL suggest the patient will have a poor response to IVF.(21) Assessment of fallopian tube patency: Hysterosalpingogram ( HSG) is performed as the first-line test for evaluation of tubal patency because of its therapeutic, as well as diagnostic, benefits.(22) Non- or minimally invasive alternatives to HSG for investigation of tubal sub fertility include Chlamydia antibody testing and/or hysterosalpingo-contrast sonography (HyCoSy) .(23) When the diagnosis is in doubt, more invasive tests can be used to confirm the diagnosis, and provide an opportunity for concurrent therapeutic intervention. These tests include laparoscopy with chromotubation . Assessment of the uterine cavity: By either hysterosalpingography or ultrasonography or three dimensional sonography or saline infusion sonography or Hysteroscopy .

Assessment of Male factor of infertility:

The causes of male infertility can be divided into four main areas(24) Hypothalamic pituitary disease (secondary hypogonadism) 1 to 2 percent Primary hypogonadism 10 to 15 percent Post-testicular defects (disorders of sperm transport) 10 to 20 percent Seminiferous tubule dysfunction 60 to 80 percent including microdeletions of the Y chromosome Evaluation of infertile male consist of: History Physical examination
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 Semen analyses Genetic tests Endocrine testing HISTORY : The evaluation of an infertile man should begin with a detailed history that focuses on potential causes of infertility. The clinician should inquire about: Developmental history, including testicular descent, pubertal development, loss of body hair, or decrease in shaving frequency . Chronic medical illness Infections, such as mumps orchitis, sinopulmonary symptoms, sexually transmitted infections, and genitourinary tract infections including prostatitis Surgical procedures involving the inguinal and scrotal areas such as vasectomy, orchiectomy, and herniorrhaphy Drugs and environmental exposures, including alcohol, radiation therapy, anabolic steroids, cytotoxic chemotherapy, drugs that cause hyperprolactinemia, and exposure to toxic chemicals (eg, pesticides, hormonal disrupters) Sexual history, including libido, frequency of intercourse, and previous fertility assessments of the man and his partner School performance, to determine if he has a history of learning disabilities suggestive of Klinefelter's syndrome PHYSICAL EXAMINATION : The physical examination should include a general medical examination with a focus on finding evidence of androgen deficiency, which may accompany decreased fertility. The clinical manifestations of androgen deficiency depend upon the age of onset. Androgen deficiency during early gestation presents as ambiguous genitalia; in late gestation as micropenis; in childhood as delayed pubertal development; and in adulthood as decreased sexual function, infertility, and eventually, loss of secondary sex characteristics. The examination of the man should include the following components. General appearance :

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Eunuchoidal proportions (upper/lower body ratio <1 with an arm span 5 cm >standing height) suggest androgen deficiency antedating puberty. On the other hand, increased body fat and decreased muscle mass suggest current androgen deficiency. Skin: Loss of pubic, axillary, and facial hair, decreased oiliness of the skin, and fine facial wrinkling suggest long-standing androgen deficiency. External genitalia : Several abnormalities that can affect fertility can be recognized by examination of the external genitalia: Incomplete sexual development can be recognized by examining the phallus and testes and finding a Tanner stage other than 5 Diseases that affect sperm maturation and transport can be detected by examination of the scrotum for absence of the vas, epididymal thickening, varicocele, and hernia. The presence of a varicocele should be confirmed with the man standing and performing a Valsalva maneuver. Decreased volume of the seminiferous tubules can be detected by measuring testicular size by Prader orchidometer or calipers. The Prader orchidometer consists of a series of plastic ellipsoids with a volume from 1 to 35 mL. In an adult man, testicular volume below 15 mL and testicular length below 3.6 cm are considered small.(25) Breasts : Gynecomastia suggests a decreased androgen to estrogen ratio. STANDARD SEMEN ANALYSIS: The semen analysis is the cornerstone of the assessment of the male partner of an infertile couple. In addition to the standard analysis, specialized analyses can be performed in some laboratories. (26) The standard semen analysis consists of the following: Measurement of semen volume and pH Microscopy for debris and agglutination Assessment of sperm concentration, motility, and morphology Sperm leukocyte count Search for immature germ cells WHO lower reference limits : The World Health Organization (WHO) has published revised lower reference limits for semen analyses.(27) The following parameters represent
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the generally accepted 5th percentile (lower reference limits and 95% confidence intervals in parentheses), derived from a study of over 1900 men whose partners had a time-to-pregnancy of 12 months.(27) Volume : 1.5 mL (95% CI 1.4-1.7) Sperm concentration : 15 million spermatozoa/mL (95% CI 12-16) Total sperm number : 39 million spermatozoa per ejaculate (95% CI 33-46) Morphology : 4 percent normal forms (95% CI 3-4), using "strict" Tygerberg method. (28) Vitality : 58 percent live (95% CI 55-63) Progressive motility : 32 percent (95% CI 31-34) Total (progressive + non progressive motility) : 40 percent (95% CI 38-42) Semen volume : The mean semen volume in the WHO study was 3.7 mL; the lower reference limit was 1.5 mL .(27) A low volume in the presence of azoospermia (no sperm) or severe oligozoospermia (severely subnormal sperm concentration) suggests genital tract obstruction (eg, congenital absence of the vas deferens and seminal vesicles or ejaculatory duct obstruction). Congenital absence of vas deferens is diagnosed by physical examination and low semen pH, whereas ejaculatory duct obstruction is diagnosed by the finding of dilated seminal vesicles on transrectal ultrasonography. Low semen volume with normal sperm concentration is most likely due to semen collection problems (loss of a portion of the ejaculate) and partial retrograde ejaculation. Androgen deficiency is also associated with low semen volume and low sperm concentration. The patient should be asked to return for a carefully collected repeat semen sample after emptying the bladder; post-ejaculation urine can be collected to assess whether there is retrograde ejaculation .(27) Sperm concentration : The lower reference limit for sperm concentration is 15 million/mL (95% CI 12-16) .(41) However, some men with sperm counts considered to be low can be fertile, while others above the lower limit of normal can be subfertile . (29-31) and, for the purposes of fertilization in vitro, 10 million/mL or even less can be satisfactory .(28)

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If no spermatozoa are seen, the semen should be centrifuged and the pellet examined for the presence of spermatozoa before the diagnosis of azoospermia is given. The presence of any sperm in the pellet will allow intracytoplasmic sperm injection (ICSI) to be performed with ejaculated spermatozoa instead of sperm collected by testicular aspiration. Round cells observed in the semen smear may be leukocytes, immature germ cells or degenerating epithelial cells .(42) Presence of immature germ cells in the semen usually indicated disorders of spermatogenesis. Leukocytes can also be seen microscopically and counted with the hemocytometer. Agglutination suggests autoimmunity, which should be confirmed by tests for sperm surface antibodies. Sperm motility: Sperm motility is assessed microscopically and is classified as progressive motility, non-progressive motility, and immotile spermatozoa. At least 40 percent of spermatozoa should be motile and at least 32 percent should have progressive motility. If sperm motility is poor, sperm vitality should be assessed by supravital stains or the hypoosmotic swelling test to determine whether the majority of immotile spermatozoa are dead .(28) The distinction between living, non-moving sperm, and dead sperm influences the type of assisted reproductive treatment that can be used for the induction of pregnancy. Sperm morphology: The criteria for normal morphology were previously based mainly on shape, as observed microscopically. They now also include length, width, width ratio, area occupied by the acrosome, and neck and tail defects. (27, 31) These criteria are called strict criteria and have good predictive value in terms of fertilization in vitro and pregnancy rates after in vitro fertilization (IVF). (31) Based upon these correlations between "strict criteria" sperm morphology and IVF pregnancy rate, the lower limit of normal sperm morphology was estimated to be about 4 percent of spermatozoa. (27,30,31) Leukocytes: White blood cells, mainly polymorphonuclear leukocytes, are frequently present in the seminal fluid. Assessment of white blood cells is usually performed by using the peroxidase stain. The peroxidase
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positive cells are counted using the hemocytometer. (27) Presence of increased white blood cells in the ejaculate may be a marker of genital infection/inflammation and may be associated with poor semen quality because of the release of reactive oxygen species from the leukocytes. The suggested cut-off for the diagnosis of a possible infection is one million leukocytes/mL of ejaculate. However, this cut-off is not evidence-based . (32) Hyperviscosity: Hyperviscosity may interfere with the semen analysis, in particular, evaluation of sperm motility. Hyperviscous samples should be treated in the laboratory to reduce viscosity by passing the sample via a large gauge needle, diluting with a physiological solution or use of enzyme digestion before testing for sperm parameters in the laboratory. Although the cause of hyperviscosity is unclear, it is thought to be due to inflammation of the genitourinary tract. (33) GENETIC TESTS : The introduction of ICSI has made it possible for men with severe oligozoospermia and azoospermia to father children, but the genetic risks of this highly invasive technique must be considered. These include the risks of transferring the cystic fibrosis conductance regulator (CFTR) gene, somatic and sex chromosome abnormalities, and microdeletions of the Y chromosome .(34) ENDOCRINE TESTS: The endocrine assessment of an infertile man includes measurements of serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), and other tests if needed: (35) Serum testosterone Measurement of a morning serum total testosterone is usually sufficient. In men with borderline values, the measurement should be repeated and measurement of serum free testosterone may be helpful.(35) Serum LH and FSH When the serum testosterone concentration is low, high serum FSH and LH concentrations indicate primary hypogonadism and values that are low or normal indicate secondary hypogonadism. (35) Men with low sperm counts and low serum LH concentrations who are wellandrogenized should be suspected of exogenous anabolic or androgenic steroid abuse. (35) Other : Serum prolactin should be measured in any man with a low serum testosterone concentration and normal to low serum LH concentration.(35) Although inhibin assays are not widely available outside of research
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laboratories, low serum inhibin concentrations may be an even more sensitive test of primary testicular dysfunction than high serum FSH concentrations, provided the assay is specific for inhibin B.(36)

OVULATION INDUCTION
Rationale for Inducing Ovulation while carrying out IUI is that ovarian stimulation has been shown to significantly improve the outcome in IUI cycles. Ovarian stimulation may improve the results of IUI by increasing the number of eggs available for fertilization and overcoming a subtle defect in ovulatory function and luteal phase. Ovarian Stimulation or Induction Patients requiring ovarian stimulation or induction can be categorized in two groups. 1. Ovulatory Patients (Ovarian Stimulation):

In these patients there is an established ovulatory pattern. Multiple studies have shown improved pregnancy rates with ovarian stimulation in these patients as compared to non-stimulated natural cycles. The aim of ovarian stimulation in ovulatory patients is to bring about multiple follicular development in order to increase the number of eggs produced & hence the number of embryos potentially available for implantation. 2. Anovulatory Patients (Ovarian Induction):

Ovulatory disorders can be identified in the woman in 18 to 25 percent of couples presenting with infertility.(2)Anovulatory patients are further divided by WHO into 3 categories: Group I: Hypogonadotrophic hypogonadism Group II: PCOS

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Group III: Ovarian failure Ovarian stimulation is aimed at achieving monofollicular development.

Drugs used for controlled ovarian hyperstimulation:1. Clomiphene Citrate (CC)

Mechanism of action: Hypothalamus and pituitary: Most evidence suggests that the primary site of clomiphene action is the hypothalamus, where it appears to bind to hypothalamic estrogen receptors, thereby blocking the negative feedback effect of circulating endogenous estradiol .(37) In vitro data suggest that clomiphene citrate also has a pituitary site of action where it causes an increase in the gonadotropin response to GnRH. (38) Clomiphene acts primarily as an antiestrogen in the uterus, cervix, and vagina. The following findings may explain the low pregnancy rates in clomiphene-induced ovulatory cycles: The normal increase in uterine volume and endometrial thickening that occurs during spontaneous menstrual cycles is largely absent during clomiphene-induced cycles, despite higher estradiol levels. (39) Abnormal luteal phase endometrial morphology has been found in some,(40) but not all, (41) studies. Clomiphene citrate directly impairs implantation efficiency in mice. (42) Data on the effect of clomiphene on cervical mucus are conflicting. While one study found no detrimental effect, (43) another noted a decrease in the quality and quantity of cervical mucus at all clomiphene doses. (44) In a metaanalysis, a detrimental effect was seen only with doses 100 mg/day. (45) Indications: Clomiphene citrate is the traditional drug of choice for ovulation induction in anovulatory infertile women with normal thyroid function, normal serum prolactin levels, and normal endogenous estrogen production, as determined by clinical observations (oligomenorrhea, estrogenic cervical mucus), a serum estradiol determination (greater than approximately 40 pg/mL), or a normal menstrual response to a progestin challenge (WHO Group II).(46) Although the
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drug also frequently is used empirically to stimulate multi-follicular development in ovulatory women with unexplained infertility (usually in combination with IUI),(47) Clomiphene Treatment Regimens Clomiphene is administered orally, typically beginning on the third to fifth day after the onset of a spontaneous or progestin-induced menses. Treatment usually starts with a single 50 mg tablet daily for a 5-day interval and, if necessary, increases by 50 mg increments in subsequent cycles until ovulation is achieved. Results of Clomiphene Treatment Clomiphene will induce ovulation successfully in 70-80 percnt; of properly selected women. (48) Among anovulatory infertile women who respond to clomiphene treatment, the overall cycle fecundability is approximately 15 percent. Monitoring of clomiphen treatment: Detection of ovulation with clomiphene using the same method to detect anovulation such as mid luteal phase progesterone, LH surge, serial ultrasonography to detect the development of follicle. hCG with clomiphen citrate In anovulatory women who fail to ovulate in response to clomiphene alone, adjuvant hCG treatment is based on the premise that clomiphene may be successful in stimulating the emergence of a preovulatory follicle but ultimately fail to trigger an endogenous LH surge and to induce ovulation. Serial transvaginal ultrasonography is required to demonstrate the phenomenon and to ensure that the ovulatory stimulus is delivered at the appropriate time. If administered blindly and prematurely, before the dominant follicle is mature enough to respond, hCG is more likely to induce atresia than ovulation. The question of when to administer hCG presents a dilemma. Although hCG commonly is administered when the lead follicle reaches 18-20 mm,(49) clinical studies indicate that the peak preovulatory follicular diameter in successful clomiphene-induced ovulatory cycles ranges between 18 and 30 mm (mean 25 mm). (50,51) Considering that the
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preovulatory follicle grows approximately 2 mm per day as it approaches maturity,(52,53) the corresponding interval may thus span up to 6 days. Normally, the preovulatory follicle triggers its own ovulatory stimulus at the peak of maturity by generating and maintaining the estrogen levels that are required to induce the LH surge. The timing of the spontaneous LH surge is therefore always optimal, but that of hCG treatment can never be more than an educated guess. Exogenous Gonadotropins Since their introduction into clinical practice in 1961, gonadotropins extracted from the urine of postmenopausal women (human menopausal gonadotropins [hMG]), in which the ratio of LH to FSH bioactivity is 1:1, have assumed a central role in ovulation induction. (54) Refinement of the initially crude preparation resulted in the availability of purified and highly purified urinary FSH. Since 1996, recombinant human FSH (rFSH, >99 percent purity) has been available. Recombinant preparations are appealing due to their ease of administration (subcutaneous rather than intramuscular), purity, and batch-to-batch consistency. Indications for Gonadotropin Treatment 1Hypogonadotropic Hypogonadism In women with hypogonadotropic hypogonadism, the drug of choice is menotropins because it contains both FSH and LH. luteal phase support with supplemental hCG (2,000-2,500 IU every 3-4 days) (55) or progesterone generally is needed to compensate for low levels of endogenous LH secretion that can prove insufficient to support normal luteal function. 2Clomiphene-Resistant Anovulation: Exogenous gonadotropins can be used intentionally to stimulate the development and ovulation of more than one mature ovum in efforts to increase cycle fecundity in older subfertile women and those with otherwise unexplained infertility; superovulation is most effective when combined with timely IUI.

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Luteal support is not required because the combined contributions of two or more corpora lutea may be reliably expected to yield supraphysiologic luteal phase serum progesterone concentrations. Treatment regmines for gonadotropins use in super ovulation 1Step up regimen : Designed to to define the effective threshold of response .In both women with hypogonadotropic hypogonadism (WHO Group I) and those with clomiphene-resistant anovulation (WHO Group II). Initial attempts to induce ovulation generally should begin with a low daily dose (75 IU daily) . After 4 to 7 days of stimulation, a serum estradiol level, with or without transvaginal ultrasonography, provides the first measure of response. Thereafter, the dose of gonadotropins may be maintained or increased, as indicated. Once the serum estradiol level begins to rise, ovarian ultrasonography to determine the number and size of developing follicles becomes essential and the frequency of evaluation increases to every 1-2 days. When the mean diameter of the lead follicle reaches 16-18 mm, hCG is administered to trigger ovum release; ovulation generally may be expected to occur approximately 36-48 hours later. In subsequent stimulation cycles, the initial dose of gonadotropins should consider the response threshold and pattern of follicular development observed in previous cycles. 2Low slow regimen Because women with PCOS often are exquisitely sensitive to low doses of gonadotropin stimulation, early and frequent monitoring generally is wise. Such women typically have a larger number of small antral follicles poised to respond to FSH stimulation (recruitable follicles).(56) Ovarian hyperstimulation, higher risks of multiple pregnancy, and the expense and frustration associated with canceled cycles usually can be avoided by using this regimen that involves low doses (37.5-75 IU daily), small increments, and a longer duration of stimulation.(57) Although most gonadotropin stimulations span an interval of 7-12 days, low-dose stimulations in women with PCOS can take longer. Insulin resistant women may be less sensitive to gonadotropin stimulation than those who are not. (58) In some such women, metformin treatment before and during gonadotropin stimulation can help to improve response, limit the number of smaller developing ovarian follicles,(59) and reduce the likelihood of cycle cancellation for excessive stimulation.(60)
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3Step-down regimen It is designed to more closely approximate the pattern of serum FSH concentrations observed in spontaneous ovulatory cycles. Treatment begins with a higher dose (150-225 IU daily) and decreases gradually thereafter in an effort to promote continued development of only the more sensitive dominant follicle while withdrawing support from the less sensitive smaller follicles in the cohort. Considering that many anovulatory women are quite sensitive to low doses of exogenous gonadotropin stimulation, the step-down method generally is best applied only after the response threshold has been established in one or more previous stimulation cycles. However, the two approaches can be effectively combined, first gradually increasing the dose of gonadotropins until a response is observed, and then decreasing the dose once a dominant follicle has emerged. Sequential clomiphen gonadotropine regimen Some clomiphene-resistant anovulatory women can benefit from sequential treatment with clomiphene and gonadotropins. The typical cycle involves a standard course of clomiphene treatment (50-100 mg daily), followed by low dose FSH or hMG (75 IU daily) beginning on the last day of clomiphene therapy or the next day; treatment is monitored and individualized thereafter as in standard gonadotropin-stimulated cycles. In most, (61,62 ) but not all studies, (63 ) cycle fecundity in sequential treatment cycles has approached or equaled that achieved with gonadotropins alone. In all, the dose and duration of gonadotropin therapy and the associated costs of monitoring were decreased significantly by 50 percnt; or more. Logically, sequential therapy generally is useful only in women who respond to clomiphene, at least to some extent. Otherwise, treatment does not effectively begin until gonadotropin therapy starts. 4Addition of GnRH agonist The elevated endogenous LH levels in many clomiphene-resistant anovulatory women with PCOS predispose to premature follicular luteinization during exogenous gonadotropin stimulation (64,65) and have been implicated as a contributing factor in the higher incidence of spontaneous miscarriage observed in those who conceive.(66) Adjuvant treatment with a long-acting GnRH agonist before exogenous gonadotropin stimulation suppresses endogenous LH levels and continued GnRH agonist treatment during gonadotropin stimulation can prevent premature luteinization. (67) The
xxii

risk that residual GnRH agonist-induced LH suppression might result in poor luteal function after ovulation induction appears more theoretical than real. (68) Monitoring Gonadotropin Therapy 1Serum Estradiol Levels To best reflect the ovarian response to stimulation and provide for an efficient flow of information, gonadotropins generally are administered in the evening, typically between 5:00 and 8:00 p.m., and serum estradiol measurements are obtained early in the morning. Results usually are available for review by midday, and new instructions regarding the dose and duration of treatment and the next scheduled evaluation are communicated before the evening dose that day is due. In general, follicles less than approximately 10 mm in mean diameter produce relatively little measurable estrogen and larger follicles secrete progressively more as they grow and approach maturity. Usually, estradiol levels rise at a constant exponential pace, doubling approximately every 2-3 days over the days before peak follicular development is achieved. A shallower or steeper slope of increase suggests the need to increase or decrease the level of stimulation. In the natural ovulatory cycle, estradiol levels peak between 200 and 400 pg/mL just before the LH surge. Comparable levels of estradiol should be expected in gonadotropin-stimulated cycles, for each mature follicle observed. Clinical judgements also must consider the number and size of smaller follicles and their lesser but collective contributions to the serum estradiol concentration. Not surprisingly, cycle fecundability increases with serum estradiol levels; unfortunately, so do the risks of multiple pregnancy and ovarian hyperstimulation. With existing gonadotropin stimulation regimens, best results generally are obtained when estradiol concentrations peak between 500 and 1500 pg/mL; pregnancies are uncommon at levels below 200 pg/mL. (69-72) 2Ultrasonography Ovarian ultrasonography defines the size and number of follicles contributing to the measured estradiol level. In the normal ovulatory cycle, the recruited cohort of antral follicles can be identified by cycle day 5-7, the dominant follicle emerges by day 8-12, grows approximately 1-3 mm per day thereafter (most rapidly over the 1-2 days immediately preceding ovulation), and measures approximately 20-24 mm in mean diameter when the LH surge occurs; lesser follicles rarely exceed approximately 14 mm in diameter. (73,74 )
xxiii

In 5-10 percnt; of spontaneous cycles, two preovulatory follicles may develop. In exogenous gonadotropin-stimulated cycles, dominant follicles exhibit a similar linear growth pattern, but reach maturity at a smaller mean diameter and over a wider range of sizes. The likelihood of ovulation increases with follicular diameter. As judged by serial ultrasonography after hCG administration, follicles 14 mm and smaller occasionaly ovulate, but about 40 percnt; of those 15-16 mm, 70 percnt; measuring 17-18 mm, 80 percent; measuring 19-20 mm in size, and virtually all larger follicles will ovulate. (75) The larger range of follicle size at maturity complicates clinical judgments. The risk of multiple gestation rises with the number of follicles likely to ovulate. Consequently, hCG generally should not be administered when the risk of multiple ovulation is high and the goal of treatment is unifollicular ovulation. A large number of intermediate and small follicles also increases risk for ovarian hyperstimulation syndrome .(76 ) Results of Gonadotropin Treatment Although exogenous gonadotropin therapy can successfully induce ovulation in over 90&percnt; of women with either hypogonadotropic hypogonadism (WHO Group I) or clomiphene resistant anovulation (WHO Group II), the pregnancy rates achieved in the two populations differ significantly.(77,78 ) In women with hypogonadotropic hypogonadism, cycle fecundity is approximately 25 percent;, equal to or even greater than that observed in normal fertile women; cumulative pregnancy rates after up to six cycles of gonadotropin stimulation approach 90 percent;. By comparison, cycle fecundity is significantly lower in clomiphene-resistant anovulatory women. Overall, cycle fecundity ranges between 5 percent; and 15 percent; and cumulative conception rates range between 30 percent; and 60 percent;; within the group, those with hyperandrogenic chronic anovulation have the poorest prognosis.(77,78) The incidence of multifetal gestation is greatly increased in pregnancies resulting from exogenous gonadotropin-induced ovulation, even in anovulatory women where the goal of treatment is unifollicular ovulation. Whereas approximately 1 in 80 (1.25 percent;) spontaneous pregnancies and 5-8 percent; of those following clomiphene treatment are multiples,(79,80 )

xxiv

approximately 15 percnt; of all pregnancies following gonadotropin-induced ovulation in ano-vulatory women are multiples. (77,7 8 ) The overall incidence of spontaneous miscarriage in gonadotropin-induced conception cycles is approximately 20-25 percent; (77, 78 ) moderately higher than is generally observed (15 percent ). As with clomiphene, there is no evidence that gonadotropin therapy is associated with any increased prevalence of congenital anomalies. (81)

INTRAUTERINE INSEMINATION
Definition: Intrauterine insemination is a technique that processes semen and separates motile, morphologically normal spermatozoa from dead sperm, leukocytes, and seminal plasma. (1) This highly motile fraction is then inserted through the cervix via a flexible or rigid catheter near the anticipated time of ovulation. (82) History Undocumented tales exist of Arabs obtaining sperm from mated mares belonging to rival groups and using the sperm to inseminate their own mares.
(83)

Leeuwenhoek (1678) and his assistant, Hamm, were the first persons to see sperm. In a letter to William Bounker of the Royal Society the Royal Society of London in which he showed a picture of sperm cells of the human and the dog. Van Leeuwenhoek described the spermatozoa as zaaddiertjes or living animalcules in human semen ... less than a millionth the size of a coarse grain of sand and with thin, undulating transparent tails. (84) He draws the conclusion that the tails must be operated by means of muscles, tendons and joints.(85) Leeuwenhoek did not have an advanced formal education, so he did not study Latin, the scientific language of the day. However, he was a clever, capable individual who ground lenses so precisely (one still exists today with 270 magnifications) that sperm were visible. (83)
xxv

More than 100 years later, in 1784, the first artificial insemination in a dog was reported by the scientist Lazzaro Spallanzani (Italian physiologist,17291799).(86) This insemination resulted in the birth of three puppys 62 days later (Belonoschkin, 1956;Zorgniotti, 1975).(87) The first documented application of AI in human was done in London in the 1770s by John Hunter,which has been called in medical history the the founder of scientific surgery. A cloth merchant with severe hypospadias was advised to collect the semen (which escaped during coitus) in a warmed syringe and inject the sample into the vagina. (83) In 1899 the first attempts to develop practical methods for artificial insemination were described by Ilya Ivanovich Ivanov (Russia, 1870-1932). (88) Although Ivanov studied artificial insemination in domestic farm animals, dogs, rabbits and poultry, he was the first to develop methods as we know today,also in human medicine.(88) The first reports on human artificial insemination originated from Guttmacher (1943), Stoughton, (89) and Kohlberg . (90,91 ) It was the real start of a new era in assisted reproduction. Phillips and Lardy (1939) were the first to use egg yolk to protect bull sperm cells from temperature shock upon cooling. This protection was explained by the effect of phospholipids and lipoproteins in the egg yolk. (92) Salisbury et al. (1941) improved the media by using egg yolk with sodium citrate, permitting the use of semen at 5 C for up to three days.(93) Polge and co-workers (1949) were the first to freeze fowl and bull spermatozoa by using glycerol in the extender media.(94) In 1953 the first successful pregnancy from artificial insemination with frozen and thawed sperm was reported, a major breakthrough in history. (83) Indications of intrauterine insemination Various clinical indications where IUI can be helpful in improving chances of conception are the following.(95) 1. Ejaculatory failure a. Anatomical (e.g. hypospadias) b. Neurological (e.g. spinal cord injury , diabetic neuropathy) c. Retrograde ejaculation ( e.g. multiple sclerosis ) 2. Psychological (e.g. impotence ) 3. Cervical Factor
xxvi

a. Cervical mucus hostility b. Poor cervical mucus 4. Mild to Moderate Male Subfertility. a. Oligospermia b. Asthenospermia c. Teratospermia d. Oligo-asthenoteratozoospermia e. Highly Viscous Semen f. Pyospermia g. Hypospermia h. Delayed Lique faction 5. Immunological factors : a. Male Antisperm Antibodies b. Female antisperm antibodies 6. Unexplained infertility 7. Endometrosis 8. Mild & Moderate with Normal Tubo Ovarian relations. 9. Ovulatory dysfunction 10.Human immunodeficiency virus (HIV)-positive male partner and HIVnegative female partner 11.Corrected Tubo-peritoneal factor 12.Combined infertility factors

Sperm Preparation for IUI There are a variety of methods for extracting sperm from the seminal plasma for IUI. The most common methods include conventional washing, the swim-up procedure, and density gradient centrifugation. The best choice among them may vary with the quality of the semen sample.(96,97) The results of a randomized study comparing the pregnancy rates achieved with IUI after a variety of sperm preparation methods suggest that swim-up and density gradient centrifugation may offer a greater chance for success than conventional sperm washing.(96) Another study found that density gradient centrifugation yielded better results than conventional
xxvii

washing when the insemination specimen contains less than approximately 20 million sperm.(97) However, a recent meta-analysis including five trials involving over 250 couples and comparing three techniques concluded that evidence is insufficient to recommend any specific preparation technique.(98) Both the conventional washing and swim-up methods allow sperm to remain in contact with dead or defective sperm and leukocytes, which produce high levels of reactive oxygen species that may cause oxidative damage to sperm membranes and motility.(99) Whereas methods more sophisticated than conventional washing or swim-up may be used to prepare sperm (density gradient centrifugation, glass wool filtration, others), and often are when preparing sperm for IVF,(100) they generally are not required for IUI. Reagents used for semen preparation are: (28) 1. BWW, Earles, Hams F-10 or human tubal fluid (HTF).supplemented preferably with human serum albumin (HSA), or serum. 2. HSA, highly purified and free from viral, bacterial and prion contamination and endotoxins. 3. HSA supplement: to 50 ml of medium add 300 mg of HSA, 1.5 mg of sodium pyruvate, 0.18 ml of sodium lactate (60% (v/v) syrup) and 100 mg of sodium bicarbonate. 4. Serum supplement: to 46 ml of medium add 4 ml of heat-inactivated (56 C for 20 minutes) clients serum, 1.5 mg of sodium pyruvate, 0.18 ml of sodium lactate (60% (v/v) syrup) and 100 mg of sodium bicarbonate. 5. Isotonic density-gradient medium: to 10 ml of 10 concentrated culture medium add 90 ml of density-gradient medium, 300 mg of HSA, 3 mg of sodium pyruvate, 0.37 ml of sodium lactate (60% (v/v) syrup) and 200 mg of sodium bicarbonate. 6. Gradient 80% (v/v): to 40 ml of isotonic gradient medium add 10 ml of supplemented medium. 7. Gradient 40% (v/v): to 20 ml of isotonic gradient medium add 30 ml of supplemented medium. Methods of semen preparation are: 1. Simple washing
xxviii

This simple washing procedure provides the highest yield of spermatozoa and is adequate if semen samples are of good quality. It is often used for preparing spermatozoa for IUI. Procedure (28) First we Mix the semen sample well .then we Dilute the entire semen sample 1 + 1 (1:2) with supplemented medium to promote removal of seminal plasma. Then the diluted suspension is transferred into multiple centrifuge tubes, with preferably not more than 3 ml per tube. Centrifugation is then done at 300500g for 510 minutes. We carefully aspirate and discard the supernatants. We resuspend the combined sperm pellets in 1 ml of supplemented medium by gentle pipetting. Another Centrifugation at 300 500g for 35 minutes is done. Then we carefully aspirate and discard the supernatant. Then we resuspend the sperm pellet, by gentle pipetting, in a volume of supplemented medium appropriate for final disposition. 2. Direct swim-up

Spermatozoa may be selected by their ability to swim out of seminal plasma and into culture medium. This is known as the swim-up technique. The semen should preferably not be diluted and centrifuged prior to swim-up, because this can result in peroxidative damage to the sperm membranes.(99) Thus, a direct swim-up of spermatozoa from semen is the preferred method for separating out motile spermatozoa .(101) The direct swim-up technique can be performed either by layering culture medium over the liquefi ed semen or by layering liquefi ed semen under the culture medium. Motile spermatozoa then swim into the culture medium. This procedure gives a lower yield of spermatozoa than washing, but selects them for their motility and is useful where the percentage of motile spermatozoa in semen is low, e.g. for IVF and ICSI. Procedure (28) First we mix the semen sample well. Then we place 1 ml of semen in a sterile 15-ml conical centrifuge tube, and gently layer 1.2 ml of supplemented medium over it. Alternatively, pipette the semen carefully under the supplemented culture medium. we then incline the tube at an angle of about 45, to increase the surface area of the semenculture medium interface, and
xxix

incubate for 1 hour at 37 C. we gently return the tube to the upright position and remove the uppermost 1 ml of medium. This will contain highly motile sperm cells. Then we dilute this with 1.52.0 ml of supplemented medium. Centrifugation at 300500g for 5 minutes is done and we discard the supernatant. Then we resuspend the sperm pellet in 0.5 ml of supplemented medium for assessment of sperm concentration, total motility and progressive motility. And the specimen may be used directly for therapeutic or research purposes. 3. Discontinuous density gradients

Discontinuous density gradients can provide the best selection of goodquality spermatozoa, giving good separation from other cell types and debris. It is easier to standardize than the swim-up technique, and thus results are more consistent. This technique is used to recover and prepare spermatozoa for use in IVF and ICSI.(28) This method uses centrifugation of seminal plasma over density gradients consisting of colloidal silica coated with silane, which separates cells by their density. In addition, motile spermatozoa swim actively through the gradient material to form a soft pellet at the bottom of the tube. A simple two-step discontinuous density-gradient preparation method is most widely applied, typically with a 40% (v/v) density top layer and an 80% (v/v) density lower layer. Sperm preparation using density gradient centrifugation usually results in a fraction of highly motile spermatozoa, free from debris, contaminating leukocytes, non-germ cells and degenerating germ cells.(28) Procedure (28) First we prepare the density-gradient medium in a test-tube by layering 1 ml of 40% (v/v) density-gradient medium over 1 ml of 80% (v/v) densitygradient medium. Then we mix the semen sample well. Then we place 1 ml of semen above the density-gradient media and centrifuge at 300400g for 15 30 minutes. Then we remove most of the supernatant from the sperm pellet. Then we resuspend the sperm pellet in 5 ml of supplemented medium by gentle pipetting (to aid removal of contaminating density-gradient medium) and centrifuge at 200g for 410 minutes. We repeat the washing procedure

xxx

.Then we resuspend the final pellet in supplemented medium by gentle pipetting so that concentration and motility can be determined. Timing of insemination For obvious reasons and for best results, IUI should be timed to coincide with the time of spontaneous or induced ovulation. Normal sperm can survive in the female reproductive tract and retain the ability to fertilize an egg for at least 3 days, but an oocyte can be successfully fertilized for only approximately 12-24 hours after it is released.(102) In normal fertile couples, the probability of conception rises progressively over an interval of 5-6 days and peaks when intercourse occurs on the day before or day of ovulation.(103,104) The longevity of normal sperm in the female genital tract relates, in part, to their retention within the cervical mucus which, of course, is bypassed by IUI. Although unproven, there is reason to believe that sperm may have a significantly shorter functional lifespan after IUI. Logically, the lower numbers and motility of infertile partner sperm may be even more limiting. Cryopreservation damages sperm (105) and even frozen-thawed donor sperm lose viability and motility more rapidly than fresh normal sperm. The timing of IUI in the treatment of male factor infertility is therefore far more critical for success than the timing of natural intercourse in infertile couples, regardless whether infertile partner sperm or frozen donor sperm are used. Generally ovulation may be expected to occur on the day before the midcycle rise in basal body temperature (BBT) (104) or 14-26 hours after the urinary LH surge is first detected. (106) In natural and clomiphene-stimulated cycles, the most practical and reliable method for timing IUI involves urinary LH monitoring beginning approximately 3 days before expected ovulation and insemination on the day following detection of the LH surge. When ovulation is triggered by injection of exogenous hCG in natural or stimulated cycles, IUI generally is best performed approximately 34-40 hours later. Technique of insemination Immediately before performing IUI, removal of any excess mucus that might clog the catheter tip is recommended. The tip of the insemination
xxxi

catheter is then simply inserted into the cervical os and advanced slowly into the uterine cavity. A large variety of specialized catheters having varying rigidity is readily available from commercial sources and any may be used. Designs involving a stiffer moldable outer sheath over a more atraumatic and flexible inner catheter are the most versatile. The insemination specimen (approximately 0.5 mL) should be introduced slowly over 10-30 seconds. Although there are no data to indicate that it matters, it is customary to have the patient remain supine for approximately 10-15 minutes after insemination. Although some have suggested that two inseminations (12 and 34 hours after hCG-induced ovulation) yield a higher cycle fecundability than a single IUI,(107) other similarly designed studies have found no such advantage.(108) A meta-analysis including three randomized controlled parallel trials involving nearly 400 couples concluded that available data do not allow a confident conclusion.(109) Two studies of cycle fecundability after therapeutic donor inseminations have observed that two inseminations are no more effective than one.(110) In our study we compare between natural ovulatory cycle with IUI versus ovulation induction with IUI in male factor of subfertility and their effect on clinical pregnancy rate.

xxxii

AIM OF THE WORK

The aim of the work is to compare between intrauterine insemination with natural ovulatory cycle and intrauterine insemination with controlled ovarian hyperstimulation in cases of male factor of infertility and its effect on clinical pregnancy rate.

xxxiii

PATIENTS

The study was carried out on forty eight women who had an intrauterine insemination recruited from EL-Shatby Maternity University Hospital.

Inclusion criteria:
Women undergone intrauterine insemination for male subfertility in case of total sperm concentration 10106/ml with motility rate type A +B 30 %.

Exclusion criteria:
Women undergone intrauterine insemination for any other reason either cervical factor of infertility or unexplained infertility. Women undergone intrauterine insemination with normal semen parameters. Women undergone intrauterine insemination and had anovulation problem. Women undergone intrauterine insemination and had abnormal tubal factor. Women undergone intrauterine insemination and had an abnormal peritoneal factor.

xxxiv

All cases signed an informed consent to declare their agreement. All patients divided randomely by computer generated randomization into two study groups: Group (): 24 women undergone intrauterine insemination subjected to insemination after natural cycle with no ovarian hyperstimulation. Group (II): 24 women undergone intrauterine insemination subjected to controlled ovarian hyperstimulation with HMG.

xxxv

METHODS

All patients were subjected to the following: Investigation workup of infertility, which include: 1- Full history and thorough physical examination. 1. Semen analysis. 2. Ovulation assessment using day 3 FSH level, antral follicle count and serial transvaginal ultrasound. 3. Evaluation of uterine morphology by transvaginal ultrasound and hysterosalpingography. 4. Evaluation of tubal patency by either hysterosalpingography or laparoscopy. In patients selected to be in group (I): Monitoring of follicular growth and endometrial development by: Serial transvaginal ultrasound, women underwent a basal transvaginal ultrasound assessment at the beginning of their menstrual period, and on the 10th day of the cycle. Patients tested their urine samples once daily between 18.00 h and 19.00 h with a urinary semi quantitative monoclonal antibody based kit with a detection level of 40 IU (Planney, Dkt, Switzerland) starting on an individually calculated cycle day for the occurrence of an endogenous LH surge. As soon as they had detected the LH surge, A single IUI was done about 24 hours after the detection of the LH peak. Time of insemination: About 24 hours after detection of LH surge using LH surge detection kit.
xxxvi

In patients selected to be in group (II) Ovulation induction by: Human menopausal gonadotropin using step down protocol using two ampoules of HMG ,Merional 75 IU (IBSA international, Switzerland), per day . Then, the dose was tailored by repeat transvaginal ultrasound. Monitoring of follicular growth and endometrial development by: Serial transvaginal ultrasound. First, day 3 ultrasound is done to exclude ovarian cyst .Then, another ultrasound done after 5 days ,if dominant follicle equals or more than 10 mm the dose is decreased to one ampoule and another transvaginal ultrasound made 3 days later .But, if dominant follicle less than 10 mm ,the same dose is continued for 3 days. Then, another ultrasound is done. Triggering of ovulation: By human chorionic gonadotropine (hCG) when the leading follicle is at least 17-18 mm. Time of insemination: About forty hours after the hCG injection. .(111) In patients of both groups: Bed rest in supine position after insemination for 15 minutes. Timed intercourse within 12-18 hours after insemination. Sperm preparation: By swim up technique. First mixing the semen sample well, then we place 1 ml of semen in a sterile 15-ml conical centrifuge tube, and gently we layer 1.2 ml of supplemented medium over it. Alternatively, pipette the semen carefully
xxxvii

under the supplemented culture medium. Then we incline the tube at an angle of about 45, to increase the surface area of the semenculture medium interface, and incubate for 1 hour at 37 C, we gently return the tube to the upright position and remove the uppermost 1 ml of medium. This will contain highly motile sperm cells, then we dilute this with 1.52.0 ml of supplemented medium, then we centrifuge at 300500g for 5 minutes and discard the supernatant, we resuspend the sperm pellet in 0.5 ml of supplemented medium for assessment of sperm concentration, total motility and progressive motility. The specimen may be used directly. The media used is Hams F-10 supplemented by with human serum albumin (HSA) Technique of insemination: Insertion of vaginal speculum, then removal of any excess mucus that might clog the catheter tip is recommended. The tip of the outer sheath of the insemination catheter is then simply inserted into the cervical os and the inner advanced slowly into the uterine cavity. The insemination specimen (approximately 0.5 mL) should be introduced slowly over 10-30 seconds. The catheter used is embryo transfer catheter set (Labotect , Germany). Measured outcome: Clinical pregnancy rate which is defined as a rising level of beta subunit of human chorionic gonadotropin( - hCG) combined with ultrasound visualization of a pulsating gestational sac.

xxxviii

RESULTS

There were no significant difference between the two studied groups according to age ,duration of infertility, and body mass index as p values were 0.18, 0.59, and 0.16 respectively.
Table (1): Comparison between the two studied groups according to demographic data Group I Age <30 30 Min. Max. Mean SD Median Duration of inferility Min. Max. Mean SD Median BMI Min. Max. Mean SD Median 20.0 30.0 24.17 2.72 23.50 20.0 26.0 23.25 1.76 23.50
t

Group II 20 (83.3%) 4 (16.7%) 21.0 38.0 27.08 4.81 26.50 1.17 7.0 2.70 1.59 2.25

Test of sig.

16 (66.7%) 8 (33.3%) 21.0 40.0 28.08 6.08 28.0 1.0 6.0 2.93 1.56 2.75

p = 0.182

p = 0.522

MW

p = 0.588

p = 0.164

p: p value for comparing between the two studied group t: Student t-test MW: Mann Whitney test 2: Chi square test

xxxix

Group I 90 80 70 60 Group II

Percentage

50 40
30

20 10 0 <30 Age 30

Figure (1):

Comparison between the two studied groups according to age

Figure (2):

Comparison between the two studied groups according to Duration of inferility

xl

30

25

Mean of BMI

20

15

10

0 Group I Group II

Figure (3):

Comparison between the two studied groups according to BMI

xli

There was no significant statistical difference between two studied Groups according day 3 FSH as p value was 0.85 .
Table (2): Comparison between the two studied groups according to day 3 FSH Group I Day 3 FSH Min. Max. Mean SD Median 5.0 11.0 7.42 1.73 7.0 5.0 10.0 7.33 1.37 7.0 0.851 Group II P

p: p value for Student t-test for comparing between the two studied group

10 9 8

Mean of day 3 FSH

7 6 5 4
3

2 1 0 Group I Group II

Figure (4):

Comparison between the two studied groups according to day 3 FSH

xlii

There were no significant statistical differences between the two studied groups according to male age and number of smokers as p value were 0.22 and 0.56 .
Table (3): Comparison between the two studied groups according to male age and smoking habit. Group I Male age Min. Max. Mean SD Median Smokers -ve +ve 12 (50%) 12 (50%) 14 (58.3%) 10 (41.7)

Group II 24.0 42.0 29.58 4.91 29.0

Test of sig.

23.0 42.0 31.42 5.45 30.0

p = 0.217

p = 0.562

p: p value for comparing between the two studied group t: Student t-test 2: Chi square test

xliii

35 30

Mean of male age (years)

25

20 15 10
5

0 Group I Group II

Figure (5):

Comparison between the two studied groups according to male age

Group I

60

Group II

50

Percentage

40

30

20

10

0 -ve Smoker +ve

Figure (6):

Comparison between the two studied groups according to smoking habit.

xliv

There was no significant statistical difference between the two studied groups regarding sperm count per ml
Table (4): Comparison between the two studied groups according sperm parameters regarding sperm count Group I Sperm Count (million/ml) Min. Max. Mean SD Median 12.0 45.0 23.83 10.70 19.0 13.0 120.0 43.33 32.38 35.0 0.018* Group II P

p: p value for Mann Whitney test for comparing between the two studied group *: Statistically significant at p 0.05

Figure (7):

Comparison between the two studied groups according sperm count

xlv

Although there was no statistical significant difference between the two studied groups according the percentage of sperm motility type (A) per ml semen ,there was statistical significant difference between the two studied groups according to percentage of sperms with motility type (B) per ml semen .as p values were 0.13, 0.001 respectively.
Table (5): Comparison between the two studied groups according to sperm sperm parameters regarding sperm motility by percentage (%) Group I Motility A (%) Min. Max. Mean SD Median Motility B (%) Min. Max. Mean SD Median 10.0 40.0 25.25 10.35 24.0 25.0 50.0 37.83 8.33 37.50 <0.001* 15.0 50.0 32.92 10.76 32.50 20.0 45.0 28.83 7.93 28.0 0.133 Group II P

p: p value for Student t-test for comparing between the two studied group *: Statistically significant at p 0.05
Group I 40 35 30 Group II

Mean of Motility

25 20 15 10 5 0 Motility A Motility B

xlvi

Figure (8):

Comparison between the two studied groups according to sperm motility by percentage (%)

xlvii

There was no significant statistical difference between the two studied groups regarding sperm morphology per ml as p value was 0.98
Table (6): Comparison between the two studied groups according to sperm morphology . Group I Abnormal forms Min. Max. Mean SD Median 50.0 90.0 76.42 10.76 79.0 50.0 95.0 76.33 14.23 76.50 0.981 Group II P

p: p value for Student t-test for comparing between the two studied group

90 80

Mean of abnormal forms

70 60 50 40 30 20 10 0 Group I Group II

Figure (9):

Comparison between the two studied groups according to sperm abnormal forms

xlviii

There was 29 percent of cases of group II (ovulation induction group ) has had one leading follicles at day of hCG and 50 percent of cases has had two follicle and 21 percent has had three leading follicles at day of hCG.
Table (7): Distribution of the studied cases of group II according to number of follicles at day of hCG No Follicle HMG 1 2 3 7 12 5 29 50 21 %

No. of follicles at day of hcg

1 2 3

17%

50%

33%

Figure (10): Distribution of the studied cases of group II according to number of follicles at day of hCG.

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There were 25 % of cases of group II (ovulation induction group) used 13 ampoules ,33 % used 14 ampoule ,25 % used 15 ampoule , and 17 % used 16 ampoule of HMG for induction of ovulation .
Table (8): Distribution of the studied cases of group II according to number of ampoules of HMG used for induction of ovulation. No Number of ampoules of HMG 13 14 15 17 6 8 6 4 25.0 33.3 25.0 16.7 %

35 30 25
20

Percentage

15 10 5 0
13 14 15 17

Number of ampoules of HMG

Figure (11): Distribution of the studied cases of group II according to number of ampoules of HMG used for induction of ovulation.

There was no statistical significant difference between the two studied groups regarding the clinical pregnancy rate as p value was 0.48
Table (9): Comparison between the two studied groups according to clinical pregnancy rate Group I No. Pregnancy -ve +ve 18 6 75.0 25.0 20 4 83.3 16.7 0.477 % Group II No. %

p: p value for Chi square test for comparing between the two studied group

90 80 70 60
Percentage

Group I Group II

50 40 30 20 10 0 -ve Pregnancy +ve

Figure (12): Comparison between the two studied groups according to clinical pregnancy rate

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DISCUSSION
Artificial insemination is one of the most common assisted reproductive technique .it has been used for more than 200 years since John Hunter in 1770 s made first insemination. One of most common indications of intrauterine insemination is mild and moderate male subfertility. The aim of our work was to compare between intrauterine insemination with natural ovulatory cycle and intrauterine insemination with controlled ovarian hyperstimulation in cases of male factor of infertility and its effect on clinical pregnancy rate. In our study forty eight patient were recruited from El Shatby Maternity university hospital between June 2012 and March 2013.All of them have had intrauterine insemination. These patients were allocated into two groups: Group (): 24 women underwent intrauterine insemination were subjected to insemination after natural cycle with no ovarian

hyperstimulation. Group (II): 24 women underwent intrauterine insemination were subjected to controlled ovarian hyperstimulation with HMG.

Our study showed that there is no statistically significant difference regarding clinical pregnancy rate between ovulation induction with intrauterine insemination group (25 %) and natural cycle intrauterine insemination group (16.7 %) .
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Goverde (112) studied pregnancy outcome of 310 natural and 334 mildly hyperstimulated cycles for IUI in 171 couples with unexplained or mild male factor subfertility was analysed on a patient level with random coefficient models. His results showed that pregnancy rates were similar: 35 % and 39.8% per couple in the natural and mildly hyperstimulated cycles respectively (P = 0.60).So, He concluded that the application of a mild hyperstimulation protocol as an alternative to a standard hyperstimulation protocol for IUI does not result in higher pregnancy rates than IUI in the natural cycle. This result is in agreement with our study although he used a larger number of patients in comparison with ours. Cohlen (113) in a randomized crossover trial that investigated whether the efficacy of IUI in natural or stimulated cycles was related to the severity of male subfertility. Seventy-four couples completed 308 treatment cycles. Thirteen pregnancies occurred after IUI in a natural cycle (pregnancy rate per completed cycle: 8.4%) and 21 after IUI in a stimulated cycle (pregnancy rate per completed cycle: 13.7%). The efficacy of IUI in stimulated cycles was related to the severity of the semen defect. In couples with a total motile sperm count less than 10106, ovarian stimulation did not improve treatment outcome, while it did in couples with a total motile sperm count more than 10106. Compared with the expected chance of conceiving spontaneously without treatment, both natural and stimulated cycles improved the probability of conception. They conclude that, for the group as a whole, ovarian stimulation did not improve the probability of conception. This result is in agreement with our study.

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Guzick (114), Who studied 932 couples in which the woman had no identifiable infertility factor and the man had motile sperm. He founded that the 231 couples in the group treated with superovulation and intrauterine insemination had a higher rate of pregnancy (33 percent) than the 234 couples in the intrauterine-insemination group (18 percent), so he concluded that treatment with induction of superovulation and intrauterine insemination is as twice as likely to result in pregnancy as is treatment with intrauterine insemination alone. this is in contrary to our study. The conflict with our study may be due to that he divided the cases into four different groups of patients with a group of a 231 couples that treated with super ovulation and IUI and a group of 234 couples treated with IUI alone and a third group of 234 couples treated with ovarian hyperstimulation and intracervical insemination and the forth group of 233 couple treated by intracervical insemination alone. This large number of groups increase the possibility of bias and he used different statistical analysis of stratified, discrete-time Cox proportional-hazards analysis thats different from ours.

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SUMMARY
Artificial insemination is one of the most common assisted reproductive technique .It has been used for more than 200 years since John Hunter in 1770 s made first insemination. One of most common indications of intrauterine insemination is mild and moderate male subfertility. The aim of our work was to compare between intrauterine insemination with natural ovulatory cycle and intrauterine insemination with controlled ovarian hyperstimulation in cases of male factor of infertility and its effect on clinical pregnancy rate. In our study forty eight patient were recruited from El Shatby Maternity university hospital between June 2012 and March 2013.All of them have had intrauterine insemination and were fulfilling the required .These patients were allocated into two groups: Group (): 24 women underwent intrauterine insemination were subjected to insemination after natural cycle with no ovarian hyperstimulation. Group (II): 24 women underwent intrauterine insemination were subjected to controlled ovarian hyperstimulation with HMG. The measured outcome was the clinical pregnancy rate . The results of this thesis can be summarized as follows:

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There is no statistically significant difference regarding clinical pregnancy rate between ovulation induction with intrauterine insemination group (25 %) and natural cycle intrauterine insemination group (16.7 %) in case of mild to moderate male factor of subfertility.

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CONCLUSION

In case of mild to moderate male factor of subfertility ,there is no statistically significant difference regarding clinical pregnancy rate between ovulation induction with intrauterine insemination group and natural cycle intrauterine insemination group .

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RECOMMENDATIONS
In case of mild to moderate male factor of subfertility ,intra uterine insemination is preferred as the first line of treatment. Natural cycle IUI is preferred in case of mild to moderate male factor of subfertlity considering the cost and benefit to the patient.

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